• An Introduction to Multiplex Immunohistochemistry - Part 1 of 4
    With the advancement of immunotherapeutics, the urge to understand the tumor microenvironment has never been more pressing. Immunohistochemistry is a powerful tool used to examine protein expression, distribution, and activation in situ. Antibodies specific to an antigen of interest are used to detect the antigen in thin sections of flash frozen or formalin-fixed paraffin-embedded tissue. Visualization of the antigen is achieved using either an enzymatic reaction that induces chromogen precipitation at the site of antibody-antigen binding, or fluorescent reporters. Fluorescent reporters may be directly conjugated to the primary antibody used to detect the antigen of interest (direct immunofluorescence), or may be attached to a secondary antibody that detects the species-specific primary antibody (indirect immunofluorescence). The latter is more common, as it achieves more sensitive antigen detection.
  • Practical Overview of Multiplex Immunohistochemistry using TSA - Part 2 of 4
    Simultaneous detection of multiple distinct proteins of interest within a single sample can be achieved with carefully optimized fluorescent multiplex immunohistochemistry (mIHC) using tyramide signal amplification (TSA). This technique utilizes an unconjugated primary antibody specific to the protein of interest, and a primary specific secondary antibody conjugated to horseradish peroxidase (HRP). Detection is achieved with the fluorophore-conjugated HRP substrate, tyramide. When activated, tyramide forms covalent bonds with the tyrosine residues on or near the protein of interest. This permanent deposition allows the primary/secondary antibody pair to be stripped from the sample without disrupting the antigen-associated fluorescence signal. Thus, multiple rounds of staining can be performed in sequence on a given sample, without fear of the crosstalk that would otherwise result from using multiple primary antibodies raised in a single species.
  • Benefits of Fluorescent Multiplex Immunohistochemistry using Tyramide Signal Amplification - Part 3 of 4
    Fluorescent multiplex immunohistochemistry (mIHC) with tyramide signal amplification (TSA) has several advantages over one-color or traditional mIHC.
  • Evolution and Future of Multiplex Immunohistochemistry using Tyramide Signal Amplification - Part 4 of 4
    Since fluorescent labels were first conjugated to antibodies against targets of interest in the 1940s, a number of landmark developments have enabled the development of immunohistochemistry. These developments include antibody purification and labeling, enzyme digestion and heat-induced approaches to epitope retrieval, signal amplification, and improvements in imaging technologies.
  • ALDH1A1: A Protein Of Many Hats
    It’s Saturday night and you’re getting a few drinks with friends. Someone suggests shots, and afterward you notice that one of your friends is bright red. “Did you get sunburnt today?” you ask.

    “It’s Asian glow!” she says, “I’m Taiwanese, so I don’t have the gene that breaks down alcohol.”
  • Albumin is a Useful Marker of Organ Function and Tissue Damage
    Bethyl’s Mouse Albumin ELISA kit is a versatile reagent with applications in several molecular biology and biomedical research fields. This is because albumin, which is normally found in the blood, can be used as an indicator of organ function and tissue damage.
  • Between Yes and No: B7-H4 and T cell Activation
    Immunotherapy has become an increasingly important therapeutic option for treating both autoimmune diseases and various cancers. Based on the two-signal hypothesis for T cell activation, the goal is to either decrease the deleterious effect of an overactive immune system as is the case with autoimmune diseases or the opposite increase the immune response to enable targeted killing of cancer cells.
  • CAR-T cells for the treatment of blood and solid tumors
    T cells with chimeric antigen receptors, more commonly known as CAR-T cells, are used in the immunotherapy of cancer. In CAR-T cell therapy, a patient’s own T cells are taken from their blood and engineered in a laboratory to express an antigen receptor that targets a specific molecule expressed by a patient’s cancer cells. CAR-T cells contain a target-specific extracellular domain fused to the internal domain of CD3-zeta, which may or may not be fused to one or more costimulatory domains. Finally, these cells are transfused back into the patient. These cancer-specific T cells are a powerful tool that track down and kill cancer cells.
  • CD247/CD3Z: The Gatekeeper of T-cell Activation
    The TCR-CD3 complex plays an essential role in the adaptive immune response. Present on the cell surface, the T-cell receptor (TCR) initiates T-cell activation by recognizing antigens bound to the major histocompatibility complex (MHC) molecule1. However, due to the TCR’s extremely sort cytoplasmic tail it is dependent on the CD3 complex for signaling, effectively making the CD3 complex the gatekeeper for T-cell activation2. While the CD3 complex consists of multiple components, CD247/CD3Z is arguably the most important due to its role in signal transduction and its potential use as a biomarker for evaluating the status of immune system3.
  • CD3E and the Fight to Save Beta Cells
    Our immune system plays an important role in protecting us against disease. Capable of both detecting and eliminating a wide variety of pathogens from viruses to parasitic worms and cells turned rogue. Unfortunately, this blessing can also be a curse when our immune system targets normal cells as if they were foreign invaders1. This is the case with type 1 diabetes where T cells, a key part of the immune system, targets and destroys beta cells in the pancreas diminishing our bodies ability to produce insulin. Fortunately, several drugs targeting CD3E, a key component of T cell activation, are being developed and investigated to treat Type 1 diabetes and other autoimmune diseases2.
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    CD68 and PD-L1: Better Together?
    Cluster of differentiation 68 (CD68) is a glycoprotein expressed primarily by macrophages that facilitates target identification by binding to tissue- or organ-specific lectins or selectins. CD68 is a macrophage panmarker, enabling identification of the entire macrophage population regardless of phenotype. M1 macrophages are proinflammatory and antitumoral, while the M2 phenotype is associated with protumoral and immunosuppressive effects1.
  • Categories of Cancer Immunotherapy
    The advent and advancement of immunotherapy is rapidly changing the standard of care of many types of cancer. Since approval of the first modern immunotherapy in 19861, dozens of immunotherapies have been approved for clinical use2.
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    Codon Bias and Heterologous Protein Expression
    Expression of proteins in a host other than the organism from which the protein is derived, or heterologous protein expression, is an important tool in biotechnology. However, sometimes your protein of interest is poorly expressed in your host of choice. Why might this happen?
  • DEAD-Box RNA Helicases
    RNA helicases, enzymes that use ATP to unwind double-stranded RNA, are critical mediators of virtually every element of RNA metabolism. The largest family of RNA helicases is the DEAD-box proteins, comprised of 37 members in humans1. First described in the late 1980s, DEAD-box proteins are now known to play diverse roles in a variety of cellular processes, including transcription, pre-mRNA splicing, ribosome biogenesis, nuclear export of RNA, translation, and RNA degradation – most often by inducing conformational changes of their RNA targets2,3. Some DEAD-box proteins target specific RNAs, while others act more generally3.
  • Development of Modern Immuno-oncology
    Immune infiltration of human tumors as well as case studies of patients in whom concomitant infection appeared to precipitate remission of otherwise incurable malignancy were reported in the early 1800s.1 Based on the observed association between streptococcal infection of the dermis (erysipelas) and remission of soft tissue sarcoma, the first clinical cancer immunotherapy involved injection of S. pyogenes into patients. Unfortunately, inducing erysipelas proved difficult, as did curing the infection once it was successfully established. As such, the treatment was modified to include both Gram positive and Gram-negative heat-killed, non-infectious bacteria. The resulting immunostimulatory properties of this mixture achieved a remarkable long-term cure rate.2